DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election without traverse of Invention I, claims 1-3, 5-7, 13-15, 18, 21-22, 25, 28, 30-32, and 39 drawn to a synthetic/recombinant nucleic acid and a recombinant microbial production cell in the response to restriction requirement filed on July 24, 2025 is acknowledged. Claims 53, 65, and 68 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Status of the Claims
Claims 1-3, 5-7, 13-15, 18, 21-22, 25, 28, 30-32, and 39 are examined on their merits
Priority
Applicant claims domestic benefit from U.S. provisional application 62/911116 filed on October 4, 2019. Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Claims 1-3, 5-7, 13-15, 18, 31-32, and 39 are entitled to the benefit of U.S. provisional application 62/911116 and are given an effective filing date of October 4, 2019.
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e) as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. 62/911116, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. The prior-filed application does not contain support for a recombinant microbial production host cell for producing a carotenoid compound comprising a coding sequence encoding a fusion enzyme comprising an oleosin polypeptide fused to a beta-carotene ketolase and/or beta-carotene hydroxylase wherein the beta-carotene hydroxylase is an algal CrtR-B, the carotenoid compound is astaxanthin or canthaxanthin, and the host cell is Yarrowia lipolytica as claimed in claims 21-22, 25, 28, and 30. Accordingly, claims 21-22, 25, 28, and 30 are not entitled to the benefit of the prior application and are given the effective filing date of the instant application: October 5, 2020.
Information Disclosure Statement
The information disclosure statement (IDS) filed on June 24, 2022 is acknowledged and has been considered by the examiner.
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Specification
The use of the term GCG Wisconsin Package in Para. [0099] and the term Sequence Analysis Software Package in Para. [00100] which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 3 and 7 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claims contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim interpretation:
Claims 3 and 7 recite amino acid sequences “having at least 80% sequence identity” to SEQ ID NOs: 1, 3, 5, 7, and 9 in claim 3 and SEQ ID NOs: 11, 14, 16, 18, 20, 22, and 24 in claim 7.
SEQ ID NOs: 1, 3, 5, 7, and 9 comprise 159, 166, 317, 578, and 351 amino acid residues respectively. SEQ ID NOs: 11, 14, 16, 18, 20, 22, and 24 comprise 546, 543, 550, 466, 470, 468, and 466 amino acid residues respectively. The broadest reasonable interpretation of the limitation “having at least 80% sequence identity” is that any sequence that differs from the respectively claimed sequence up to 20% can read on the claimed sequence.
The instant specification is silent as to specific sequence modifications. Therefore, in SEQ ID NOs: 1 and 3 for example, 20% of the approximately 160 amino acid residues means approximately 32 amino acid residues can vary within the given sequence. Each varied amino acid residue has 19 possible choices which is a total number of approximately 1932 possible variants for the amino acid sequences of SEQ ID NOs 1 and 3. The number of possible variants for SEQ ID NOs: 5, 7, 9, 11, 14, 16, 18, 20, 22, and 24 would be larger as those sequences contain more amino acids. The number of variants as instantly claimed are greater than this example, as the substitutions are not restricted to any specific position within the sequence and any amino acid at any position can be changed to any of the other 19 possible amino acids.
Teachings of the Instant Specification:
As described above, the claims recite sequences at least 80% identical to the respective SEQ ID NO:, which includes a large number of variants that would read on the claims. The instant specification teaches various tools that can be used to analyze sequence similarity and identity scoring including use of homology algorithms in order to determine optimal alignment and tools to determine sequence identity in a general way, but does not provide any specific guidance relating to which structural changes of amino acids at which positions in which SEQ ID NOs: are able to be varied while still maintaining function as either a lipid body compartmentalization signal tag (SEQ ID NOs: 1, 3, 5, 7, and 9) or an enzyme having carotenoid cleavage activity (SEQ ID NOs: 11, 14, 16, 18, 20, 22, and 24). The instant specification also discloses that sequences with identities of 25% or higher imply similarity of function while identities of 18-25% implies similarity of structure but not necessarily function and states that “two completely unrelated or random sequences (that are greater than 100 residues) can have higher than 20% identity” (Para. [00104]) thus supporting that similar structural sequences do not necessarily retain similar functions.
The State of the Art:
Bromberg et al. (2009. Correlating protein function and stability through the analysis of single amino acid substitutions. BMC bioinformatics, 10(Suppl 8), S8) teaches that single amino acid substitutions by non-synonymous single nucleotide polymorphisms frequently disrupt protein function by altering protein structure or stability and can often affect functional binding sites (Abstract).
Sui et al. (2016. Key residues for catalytic function and metal coordination in a carotenoid cleavage dioxygenase. J. of Biological Chem., 291(37), 19401-19412) teaches that modification of single amino acid residues in an bacterial apocarotenoid-cleavage oxygenase, a carotenoid cleavage dioxgenase, can have detrimental effects on catalytic function (Abstract, Table 2, Figure 4) as well as the structure of the enzyme (Abstract, Figure 5) with many substitutions causing reduced reaction rate and enzymatic efficiency.
Abell et al. (1997. Role of the proline knot motif in oleosin endoplasmic reticulum topology and oil body targeting. The Plant Cell, 9(8), 1481-1493) teaches that substitution of the three proline residues within the proline knot motif of an oleosin resulted in an oleosin variant which failed to target to oil bodies indicating that substitution of just three amino acids, resulting in loss of the proline knot, can eliminate oleosin function (Abstract).
Analysis:
The level of skill in the art is such that one having ordinary skill would not be able to identify without undue experimentation which structural amino acid variations of the instantly claimed sequences could be made while maintaining the desired function. Thus, the outcome of substitution of up to 20% of amino acids within a sequence with any alternate amino acid at any position is highly unpredictable and those of ordinary skill in the art would not conclude that the applicants were in possession of the claimed genus of lipid body compartmentalization signal tag based on the disclosure of SEQ ID NOs: 1, 3, 5, 7, and 9 (claim 3) and the genus of proteins having carotenoid cleavage activity based on the disclosure of SEQ ID NOs: 11, 14, 16, 18, 20, 22, and 24 (claim 5).
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 1 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites the limitation “a first domain capable of functioning as a lipid body compartmentalization signal tag” in lines 3-4. After review of the prior art, the term “lipid body compartmentalization signal tag” does not appear to be a term of the art and, while the specification does provide exemplary embodiments which can function as a “lipid body compartmentalization signal tag”, the specification does not provide a definition for the term, thus rendering the scope of the claim indefinite. Appropriate correction is required.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-3, 5-7, 15, 18, 31-32 and 39 are rejected under 35 U.S.C. 103 as being unpatentable over Trumper et al. (US 2019/0194699 A1, hereafter “Trumper” in view of Kinney et al. (US 7,256,014, hereafter “Kinney”), Uniprot (B6SHN1, oleosin, Zea mays (Maize), https://www.uniprot.org/uniprotkb/B6SHN1/entry#sequences), NCBI Basic Local Alignment Search Tool (NCBI BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi#), Lopez et al. (2015. Production of β-ionone by combined expression of carotenogenic and plant CCD1 genes in Saccharomyces cerevisiae . Microb Cell Fact 14, 84, hereafter “Lopez”) and Simkin et al. (2004. Circadian Regulation of the PhCCD1 Carotenoid Cleavage Dioxygenase Controls Emission of β-Ionone, a Fragrance Volatile of Petunia Flowers, Plant Physiology, 136(3), 3504–3514, hereafter “Simkin”).
Claims 21-22, 25, and 28-29 are rejected under 35 U.S.C. 103 as being unpatentable over Kinney (as above) in view of Bailey et al. (US 2014/0234928 A1) and Tan et al. (2007. Expression of β-carotene hydroxylase gene (crtR-B) from the green alga Haematococcus pluvialis in chloroplasts of Chlamydomonas reinhardtii. J. of Applied Phycology, 19(4), 347-355.
Claim 1 recites the limitation “a first domain capable of functioning as a lipid body compartmentalization signal tag” in lines 3-4. The term “lipid body compartmentalization signal tag” does not appear to be a term of the art and the specification does not provide a definition for the term. For the purposes of examination, the term lipid body compartmentalization signal tag is interpreted to refer to a component possessing a structure or function which indicates association with lipid bodies or the lipid bilayer and which includes exemplary embodiments which are disclosed in the specification as being able to function as a lipid body compartmentalization signal tag: a lipid body structural protein, a lipid synthesis enzyme, a membrane protein, and a carotenoid-binding protein.
With regard to claim 1, Trumper teaches synthesis of an ionone compound (α-ionone) in genetically modified strain of S. cerevisae, which is considered to read on a recombinant microbial production host cell (Para. [0003]) wherein the production host cell comprises a nucleic acid sequence encoding a carotenoid cleavage dioxygenase, which is considered to read on a domain which has carotenoid cleavage activity. (Para. [0015]). Additionally, Trumper teaches that α-ionone is an apocarotenoid compound, a subclass of isoprenoids (Para. [0007], line 1), which are natural aromatic compounds produced by enzymatic cleavage of carotenoids (Para. [0011], lines 2-5) and which are frequently used in the flavoring industry (Para. [0007], line 2). Trumper teaches use of heterologous introduction of the enzymes comprising geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, lycopene cyclase, and carotenoid cleavage dioxygenase which are required for α-ionone synthesis (Abstract). Further, Trumper teaches that α-ionone is widely used in fragrance products and in as flavoring in the food industry (Para. [0007], lines 13-16), and, although α-ionone can be naturally produced by plants, naturally-produced concentrations are low and extraction and purification is expensive. Thus, most α-ionone is produced synthetically (Para. [0008], lines 1-4). Trumper also teaches that, during α-ionone synthesis, the accumulation of carotenoids is detrimental for host cell growth and that increased enzymatic efficiency is crucial for both cellular detoxification and α-ionone synthesis (Para. [0061]).
Trumper does not teach wherein the nucleic acid encodes a fusion enzyme comprising a “lipid body compartmentalization signal tag” fused to the carotenoid cleavage activity domain.
Kinney teaches a recombinant microbial production host for the production of hydrophobic/lipophilic compounds, wherein the hydrophobic/lipophilic compound is a carotenoid (Col. 2, lines 15-17), comprising an intracellular system for production of the compound and at least one genetic construct encoding an oleosin polypeptide (Col. 3, lines 24-29). Kinney teaches that oleosins are involved in lipid body storage (Col. 3, lines 6-7), thus oleosin is considered to read on domain capable of functioning as lipid body compartmentalization signal tag, and wherein the microbial production host is a bacteria, yeast, or algae (Col. 3, line 34). Additionally, Kinney teaches that carotenoids associate/aggregate in lipid monolayers and bilayers (Col. 2, lines 40-42), accumulation of carotenoids leads to changes in membrane structure which result in loss of cell viability, and that the accumulation of carotenoids is limited to the internal storage capacity of the recombinant microorganism used in production (Col. 2, lines 20-29). Further, Kinney teaches that oleosins are involved in lipid body storage (Col. 3, lines 6-7) and that introduction of a oleosin gene can be used to increase the recombinant host cell’s storage capacity of hydrophobic/lipophilic compounds such as carotenoids (Col. 3, lines 7-10). Kinney also teaches that carotenoids belong to the class of isoprenoid compounds (Col. 17, line 46) and that the “intracellular system” for production of carotenoids comprises introduction of gene construct encoding enzymes required for carotenoid synthesis including geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene dehydrogenase, and lycopene cyclase (Col. 18, lines 30-40).
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of claimed invention, to combine the recombinant host cell comprising a genetic construct encoding the oleosin polypeptide used to increase storage capacity of carotenoids and comprising constructs encoding enzymes for carotenoid synthesis (i.e., geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene dehydrogenase, and lycopene cyclase) as taught by Kinney with the recombinant host cell for producing α-ionone comprising constructs encoding enzymes for ionone synthesis (i.e., geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, lycopene cyclase) including a domain capable of having carotenoid cleavage activity (i.e. carotenoid cleavage dioxygenase) as taught by Trumper. Trumper teaches that α-ionones are produced from carotenoids (Para. [0011], lines 2-5) and have widespread usage in the fragrance and flavoring industry but that accumulation of carotenoids in production host cells is detrimental for cellular growth (Para. [0061]). Kinney teaches that introduction of an oleosin gene can be used to increase the production host’s cellular storage capacity of carotenoids (Col. 3, lines 7-10) thus reducing negative effects on host cells (Col. 2, lines 20-29). Thus, a skilled artisan would have been motivated to combine introduction of an oleosin gene as taught by Kinney with the ionone-producing recombinant host cell comprising carotenoid cleavage activity as taught by Trumper in order to increase the carotenoid (an α-ionone precursor) storage capacity in the host cell while maintaining cell viability thereby increasing the host cell capacity for α-ionone synthesis. One having ordinary skill in the art would have had a reasonable expectation of success as both Trumper and Kinney teach production of carotenoid-derived compounds using recombinant microbial host cells comprising constructs encoding carotenoid synthesis enzymes.
Neither Trumper nor Kinney teach fusion of the construct encoding the oleosin (a first domain) with the other enzymatic cellular machinery used to generate carotenoids (e.g., carotenoid cleavage dioxygenase, a second domain). However, Kinney teaches wherein the genes encoding enzymes required for β-carotene synthesis were “joined” (Example 7) and references use of “standard recombinant DNA and molecular cloning techniques well known in the art” as described by Experiments with Gene Fusions (Col. 16, lines 5-6 and 11-12), thus providing support for use of nucleic acid constructs which have been fused.
Therefore it would have been obvious, for one having ordinary skill in the art, before the effecting filing date of the claimed invention, to generate a nucleic acid construct comprising an oleosin domain (i.e., a lipid body compartmentalization signal tag) as taught by Kinney fused to a carotenoid cleavage dioxygenase domain (i.e., domain having carotenoid cleavage activity) as taught by Trumper based on their combined teachings as detailed above. As Kinney teaches wherein constructs encoding enzymes required for β-carotene synthesis were joined, a skilled artisan would have recognized that nucleic acid fusion could be applied to the oleosin and carotenoid cleavage dioxygenase domains with the predictable result of generating a nucleic acid construct encoding a fusion enzyme comprising a domain capable of functioning as a lipid body compartmentalization signal tag and domain having carotenoid cleavage activity. As Kinney teaches that carotenoids are aggregated inside lipid layers (Col. 2, lines 40-4), a skilled artisan would have been motivated to do this in order to create a construct wherein the oleosin segment would transport the ionone synthesis enzyme to the location wherein the enzyme’s substrate was naturally aggregating. On having ordinary skill in the art would have had a reasonable expectation of success as both Kinney and Trumper teach synthesis of carotenoid-based compounds using recombinant microbial host cells comprising constructs encoding carotenoid synthesis enzymes.
With regard to claim 2, Kinney teaches use genetic construct encoding an oleosin polypeptide (Col. 3, lines 28-29) and that oleosin is involved in the storage of compounds in lipid bodies (Col. 3, lines 6-7), which is considered to reasonably read on a lipid body structural protein.
With regard to claim 3, Kinney teaches use of an oleosin polypeptide wherein the any plant oleosin can be used for recombinant expression in a host cell (Col. 16, lines 52-55) and an embodiment comprising the oleosin from Zea mays with a sequence according to GENBANK accession number P21641 (Table 8).
Kinney does not teach a Zea mays construct comprising instantly claimed SEQ ID NO: 1
Instantly claimed SEQ ID NO: 1 comprises 100% sequence identity to Zea mays (corn) oleosin as taught by Uniprot entry B6SHN1.
Therefore, it would have been obvious, to one having ordinary skill in the art, before the effective filing date of the claimed invention, to substitute the Zea mays oleosin sequence as taught by Kinney, i.e., GENBANK accession number P21641 with the Zea mays oleosin sequence as taught by Uniprot entry B6SHN1 with the predictable result of generating a construct which encodes an oleosin protein (which is considered to read on a lipid body compartmentalization signal tag). One having ordinary skill in the art would have had a reasonable expectation of success as Kinney teaches that any plant oleosin can be used and a specific embodiment of a Zea mays oleosin and Uniprot teaches B6SHN1 is sequence encoding a Zea mays oleosin.
With regard to claim 5, Kinney teaches use of an oleosin polypeptide wherein the any plant oleosin can be used for recombinant expression in a host cell (Col. 16, lines 52-55) and an embodiment wherein the oleosin is defined as an protein that “includes a 60-80 amino acid-long fragment that can be aligned with the sequence segment of the Corn Oleosin Zm-II (GENBANK accession number P21641) segment extending from position 50 to 120, having more than 25% amino acid identity over that segment and sharing 8 or more of the 13 conserved amino acids listed in Table 9” (Col. 16, lines 56-62).
Kinney does not specifically teach instantly claimed SEQ ID NO: 29.
However, sequence alignment analysis using NCBI BLAST shows that instantly claimed SEQ ID NO: 29 is an 80 amino acid long fragment of a Zea mays (corn) oleosin having more than 25% amino acid identity with the segment extended from position 50 to 120 of Corn Oleosin Zm-II (GENBANK accession number P21641) and comprising 9 of the 13 conserved amino acids taught by Kinney in Table 9 (specifically at positions 77, 82, 87, 88, 89, 93, 94, 109, and 112).
Therefore, it would have obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to substitute the oleosin comprising a 60-80 amino acid-long fragment aligned with the sequence segment of the Corn Oleosin Zm-II (GENBANK accession number P21641) segment extending from position 50 to 120, having more than 25% amino acid identity over that segment, and sharing 8 or more of the 13 conserved amino acids as taught by Kinney with the corn oleosin of instantly claimed SEQ ID NO: 29 which comprises 53% sequence identity with the segment extending from position 50 to 120 of Corn Oleosin Zm-II (GENBANK accession number P21641) and 9 of the 13 conserved amino acids with the predictable result of generating a construct encoding a corn oleosin capable of being used as a lipid body compartmentalization signal tag. One having ordinary skill in the art would have had a reasonable expectation of success as Kinney teaches wherein an oleosin is defined as having greater than 25% sequence identity across the segment from positions 50 to 120 of Corn Oleosin Zm-II and 8 or more of the conserved amino acids.
With regard to claims 6 and 7, Trumper teaches a carotenoid cleavage dioxygenase (Para. [0015]) wherein the amino acid sequence of the carotenoid cleavage dioxygenase is SEQ ID NO: 10 (Para. [0029]). SEQ ID NO: 10 as taught by Trumper comprises 100% sequence identity to instantly claimed SEQ ID NO: 11.
With regard to claims 13 and 14, Trumper teaches production of α-ionone in a recombinant host cell comprising heterologous gene constructs for phytoene synthase (Para. [0042], line 2) and phytoene desaturase (CrtI) (Para. [0043], line 1), which is considered to reasonably read on phytoene dehydrogenase. Trumper teaches that α-ionone is synthesized by enzymatic cleavage of carotenoids, including by steps catalyzed by phytoene synthase and phytoene desaturase, and wherein a downstream carotenoid cleavage dioxygenase is required to complete the synthesis of α-ionone (Para. [0011]), suggesting that the action of phytoene synthase and phytoene desaturase occur in the carotenoid cleavage pathway prior to α-ionone production. Trumper is silent as to the production of β-ionone. However, one having ordinary skill in the art would recognize that α-ionone and β-ionone share a synthesis pathway and that the recombinant host cell comprising gene constructs for phytoene synthase and phytoene desaturase as taught by Trumper could also be utilized for β-ionone production. This is evidenced by the teachings of Lopez, wherein a recombinant yeast host cell comprising phytoene synthase, phytoene desaturase, and a carotenoid cleavage dioxygenase was used to produce β-ionone (Figure 1). The shared synthesis pathway of α-ionone and β-ionone is further supported by FIG. 2 in the instant specification.
With regard to claim 15, Trumper teaches recombinant yeast cells which overexpress “native nucleic acids or modified versions thereof which encode at least one enzyme of mevalonate pathway” (Claim 1a).
With regard to claim 18, Trumper teaches wherein the host cell is S. cerevisiae, i.e., a yeast (Para. [0003], line 4).
With regard to claim 21, Kinney teaches a recombinant microbial host cell for producing a hydrophobic compound (Col. 3, lines 24-25) wherein the hydrophobic compound can be a carotenoid (Col. 2, lines 15-17) and wherein the recombinant microbial host cell also comprises a genetic construct encoding an oleosin polypeptide (Col. 3, lines 28-29) and a β-carotene ketolase (Example 5). Kinney does not directly teach wherein the construct encoding the oleosin polypeptide is fused to β-carotene ketolase. However, Kinney does teach wherein the genes required for β-carotene synthesis were “joined” (Example 7) and references use of standard recombinant DNA and molecular cloning techniques well known in the art as described by Experiments with Gene Fusions (Col. 16, lines 5-6 and 11-12), thus providing support for use of nucleic acid constructs which have been fused. Further, Kinney teaches that
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to generate a nucleic acid construct wherein the oleosin polypeptide encoding sequence is fused to the β-carotene ketolase encoding sequence. As Kinney teaches a recombinant host cell comprising both an oleosin polypeptide encoding sequence and β-carotene ketolase encoding sequence are expressed and teaches fusion of sequences required for β-carotene synthesis, a skilled artisan would have recognized that the sequence encoding the oleosin polypeptide and the sequence encoding the β-carotene ketolase could be joined with the predictable result of generating a fusion enzyme. As Kinney teaches that carotenoids are aggregated inside lipid layers (Col. 2, lines 40-4), a skilled artisan would have been motivated to do this in order to create a construct wherein the oleosin polypeptide would transport the β-carotene synthesis enzyme to the location wherein the enzyme’s substrate was naturally aggregating. One having ordinary skill would have had a reasonable expectation of success as Kinney teaches both fusion of nucleic acid sequences related to β-carotene synthesis and insertion of sequences encoding an oleosin polypeptide in recombinant microbial host cells.
With regard to claim 22, as detailed above in claim 21, Kinney teaches a recombinant microbial host cell comprising an oleosin polypeptide (Col. 3, lines 28-29) and β-carotene ketolase (crtW) (Example 5) and provides support for generation of fused nucleic acid constructs.
While Kinney teaches a β-carotene hydroxylase enzyme (crtZ) for conversion of β-carotene or canthaxanthin to astaxanthin (Col. 9, lines 60 and 64-66), Kinney does not teach a host cell comprising a second coding sequence encoding a second fusion enzyme comprising an oleosin polypeptide fused to a β-carotene hydroxylase.
Bailey teaches a system for production of carotenoids in oleaginous organisms wherein the produced carotenoids are sequestered in lipid bodies (Para. [0006], lines 4-5 and 8-9) and wherein the oleaginous organism can be a recombinant fungus (Para. [0011], line 2) which produces at least one carotenoid (Para. [0011], lines 4-5). Bailey teaches wherein Y. lipolytica produce β-carotene via being engineered to express carotenoid hydroxylase, carotenoid ketolase, or both (Para. [0192]). Additionally, Bailey teaches wherein Y. lipolytica is engineered to express at least one carotenoid ketolase (crtO/crtW) in combination with at least one carotenoid hydroxylase (crtZ) (Para. [0198], lines 2-5) and embodiments wherein the carotenoid ketolase and carotenoid hydroxylase are encoded by nucleic acid sequences in the same nucleic acid molecule (Para. [0198], lines 9-12). Further, Bailey teaches wherein the carotenoid ketolase (crtO/crtW) and carotenoid hydroxylase (crtZ) polypeptides are expressed as a fusion protein (Para. [0199], lines 14), thus providing additional support for fusion constructs.
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to combine a recombinant host cell comprising the nucleic acid construct encoding an oleosin polypeptide fused to a β-carotene ketolase as taught by Kinney (as detailed above) with a recombinant host cell for production of β-carotene which is engineered to express both carotenoid ketolase and carotenoid hydroxylase as taught by Bailey. As Kinney teaches that addition of a construct encoding an oleosin polypeptide provides benefits of increasing the recombinant host cell’s storage capacity of carotenoids (Col. 3, lines 7-10) while protecting cells viability (Col. 2, lines 20-29) and Bailey teaches that both carotenoid ketolase and carotenoid hydroxylase can be expressed in the same β-carotene-producing recombinant host cell, a skilled artisan would have recognized that in addition to Kinney’s recombinant host cell comprising a nucleic acid construct encoding an oleosin polypeptide and a β-carotene ketolase, a second nucleic acid construct encoding an oleosin polypeptide fused to a β-carotene hydroxylase could be added to the recombinant host cell with the predictable result of generating a host cell comprising a first coding sequence comprising an oleosin polypeptide fused to a β-carotene ketolase and a second coding sequence encoding an oleosin polypeptide fused to β-carotene hydroxylase. One having ordinary skill in the art would have been motivated to make this combination because, as detailed above, Kinney’s teachings lead to generation of a construct that associates carotenoid synthesis enzymes with their substrates in lipid layers, thus increasing efficiency while Bailey teaches that expression of both β-carotene ketolase and β-carotene hydroxylase in a host cell can be used to produce astaxanthin, which is widely used in salmon aquaculture to provide orange coloration of wild salmon (Para. [0004], lines 5-6). A skilled artisan would have had a reasonable expectation of success as both Kinney and Bailey teach production of carotenoid compounds using recombinant microbial host cells.
With regard to claim 25, as detailed above in claims 21 and 22, the combined teachings of Kinney and Bailey teach a recombinant host cell for production of carotenoids comprising a nucleic acid construct encoding an oleosin fused to a β-carotene ketolase and a nucleic acid construct encoding an oleosin fused to a β-carotene hydroxylase.
While both Kinney and Bailey teach wherein β-carotene hydroxylase is a CrtZ, Kinney teaches wherein the recombinant production host cell is an algae (claim 2) including “Haemotacoccus” (claim 5), and Bailey teaches algae as a “source organism” for carotenoid biosynthesis polypeptide sequences (Para. [0154], lines 12-15), neither Kinney nor Bailey directly teach wherein the β-carotene hydroxylase is an algal CrtR-B.
Tan teaches a carotenoid gene (crtR-B) from green alga Haemotacoccus pluvialis encoding β-carotene hydroxylase for catalyzing the conversion of β-carotene to zeaxanthin and of canthaxanthin to astaxanthin (Abstract).
Therefore, one having ordinary skill in the art, before the effective filing date of the claimed invention, would have recognized that the β-carotene hydroxylase (CrtZ) for conversion of β-carotene or canthaxanthin to astaxanthin as taught by the combined teachings of Kinney and Bailey could be substituted with the β-carotene hydroxylase (crtR-B) from algae as taught by Tan with the predicted result of generating a construct encoding an algal CrtR-B β-carotene hydroxylase capable of producing astaxanthin. One having ordinary skill in the art would have had a reasonable expectation of success as both the combined teachings of Kinney and Bailey and Tan teach use of constructs encoding β-carotene hydroxylase for the production of astaxanthin.
With regard to claim 28, Kinney teaches wherein the carotenoid can be canthaxanthin or astaxanthin (Col. 17, lines 48-49).
With regard to claim 30, Bailey teaches wherein the recombinant fungus (i.e., the host cell) is Yarrowia lipolytica (Para. [0014], line 31).
With regard to claims 31-32 and 39, as detailed above in claim 1, Trumper teaches a use of a nucleic acid sequence encoding a carotenoid cleavage dioxygenase (claim 13) in a recombinant host cell and Kinney teaches use of a nucleic acid encoding oleosin, which is considered to read on a lipid body compartmentalization signal tag, (Col. 17, line 17) in a recombinant host cell and the combined teachings of Trumper and Kinney teach wherein the sequence encoding oleosin can be fused with the carotenoid cleavage dioxygenase thus resulting in a fusion enzyme. Additionally, Kinney teaches that oleosin is involved in storage of compounds inside lipid bodies (Col. 3, lines 6-7), which is considered to read on a lipid body structural protein. Trumper is silent as to the carotenoid cleavage dioxygenase having 9,10(9',10')-cleavage activity. However, Trumper teaches wherein the carotenoid cleavage dioxygenase is from Petunia hybrida (Ph-CCD1) (Abstract). Ph-CCD1 is a 9,10(9',10') carotenoid cleavage dioxygenase as evidenced by Simkin (Abstract).
Conclusion
No claims are allowed.
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/ERIN V PAULUS/Examiner, Art Unit 1631
/JAMES D SCHULTZ/Supervisory Patent Examiner, Art Unit 1631